Alterations in the contractile state of smooth muscle (SM) plays a key role in gastrointestinal diseases such as gastric, intestinal and sphincter dysfunction, abnormal motility and other pathologies. SM is critically modulated by a complex network of signaling pathways that regulate contractility through phosphorylation of myosin. Myosin light chain phosphatase (MLCP) is a major downstream target of these signaling pathways yet the molecular mechanisms responsible for its inhibition and activation are poorly understood and this is the focus of our proposal. The effector of RhoA GTPase, Rho-kinase (ROCK) and other kinases, phosphorylate MLCP targeting subunit (MYPT1), at Thr696 and Thr853 and inhibit MLCP, while cAMP/cGMP signals and the cyclic nucleotide target, telokin, reverse the inhibitory effect, causing GI SM relaxation. Our collaboration has lead to a novel model for the mechanism underlying the inhibition of MLCP activity upon MYPT1 phosphorylation. In the model, the segment including the phosphorylated MYPT1 at Thr696 or Thr853 directly binds to the active site of MLCP, resulting in an autoinhibition of MLCP. In Aim 1 we will determine the structure / function relationship of MYPT1 autoinhibitory (AI) domains including Thr696 (AI-1) or Thr853 (AI-2) in the regulation of gut SM tone to now rigorously test this model. The model will be validated in live fundus SM cells using FRET biosensors. The second aim will address how cAMP/cGMP signals alter MLCP activity to eliminate RhoA-mediated Ca2+ sensitization resulting in relaxation. Cyclic nucleotides are well established as physiologically important mediators of relaxation in GI SM. A major cyclic nucleotide target shown to activate MLCP is telokin, which is most highly expressed in GI SM. The pCa-force relationship is left shifted in telokin knockout mice compared to wild type. In Aim 2a we will determine the molecular mechanism(s) of telokin- induced activation of MLCP activity. Surface plasmon resonance, isothermal calorimetry, GST-pull down, proximity ligation assays (PLA) and a FRET biosensor will be used to test two molecular models of telokin- induced activation of MLCP. Functional assays will utilize GI SM from telokin -/- and WT mice. In Aim 2b we will test the hypothesis that cyclic nucleotide-induced de-autoinhibition of MLCP in different gastrointestinal smooth muscles is mediated by multiple, but dominated by different pathways to relax Ca2+ sensitized force. We will determine using photolysis of caged nucleotides, whether non-telokin mediated attenuation of the autoinhibition by cyclic nucleotides occurs through down regulation of RhoA activity by Epac activation of Rap1, through phosphorylation of Ser695 of MYPT1 or inhibitory phosphorylation of RhoA. Using our in vivo and in vitro data for simulations and fitting we expect to establish the magnitudes and hierarchy of the contribution of these signaling pathways and arrive at new mechanistic computational models of cyclic nucleotide-induced relaxation in GI SM. We expect that fundus and ileum SM will be dominated by different pathways, reflecting their different functional roles. Findings should lead to new insights for targeting therapies. PUBLIC HEALTH RELEVANCE: Diseases such as g by abnormal contraction and relaxation of smooth muscle tissues gastric, intestinal and sphincter dysfunction, dyspepsia, intestinal bowel disease, surgery- induced decreased gut motility, hypertension, cerebral and coronary vasospasm, erectile dysfunction, and bronchial asthma, among other diseases are caused by abnormal contraction and relaxation of smooth muscle tissues. We are studying the role of specific proteins, which through complex signaling pathways regulate the contractile machinery in gastrointestinal smooth muscle cells. This contractile machinery also functions in cell migration, such as occurs during development of the gastrointestinal tract and in tumor metastasis. The results of the research should translate into novel treatments for targeting these diseases.